LOCUS       sequence-209472-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression of the construct alpha-Synuclein-GFP, 
            which contains N-terminal tags, which can be cleaved later: His-tag 
            for purification and SUMO-tag for better solubility.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  AUTHORS   Afitska K, Fucikova A, Shvadchak VV, Yushchenko DA
  TITLE     Modification of C Terminus Provides New Insights into the Mechanism 
            of alpha-Synuclein Aggregation.
  JOURNAL   Biophys J. 2017 Nov 21;113(10):2182-2191. doi: 
            10.1016/j.bpj.2017.08.027. Epub 2017 Sep 20.
  PUBMED    28939194
REFERENCE   2  (bases 1 to 7019)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.bpj.2017.08.027"; journalName: "Biophys J"; date: 
            "2017-11-21- 21"; volume: "113"; issue: "10"; pages: "2182-2191"
COMMENT     Sequence Label: 1a. pETM11-SUMO-SNCA-GFP
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    43..67
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             82..104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             117..134
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             165..185
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(891..893,894..1607)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(891..893,894..1604)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(933..954)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1194..1213)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1541..1562
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1907..1924
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1977..1995)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1991..2038
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2075..2530
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2162..2181)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2372..2393
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2622..3437)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2622..3437)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3345..3364
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3429..3448
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3559..4147
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4048..4067
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4301..4318
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4333..4472
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4398..4417)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4558..4580
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4574..4765)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5574..6656)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6618..6637
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6657..6734)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6940..6959
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
       61 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgaaacatc
      121 accatcacca tcaccccatg agcgattacg acatccccac tactgagaat ctttattttc
      181 agggcgccat gggcaacgat cacattaacc tgaaagtggc cggtcaagac ggtagcgtag
      241 tccagtttaa aatcaaacgc cacacccctc tgtcgaaact gatgaaagcc tattgtgaac
      301 gccaaggtct gtctatgcgt cagatccgtt ttcgcttcga tggacagccg attaacgaaa
      361 ccgacactcc agcacagctg gaaatggaag atgaggacac cattgacgtg ttccagcaac
      421 agaccggtgg atccatggat gtattcatga aaggactttc aaaggccaag gagggagttg
      481 tggctgctgc tgagaaaacc aaacagggtg tggcagaagc agcaggaaag acaaaagagg
      541 gtgttctcta tgtaggctcc aaaaccaagg agggagtggt gcatggtgtg gcaacagtgg
      601 ctgagaagac caaagagcaa gtgacaaatg ttggaggagc agtggtgacg ggtgtgacag
      661 cagtagccca gaagacagtg gagggagcag ggagcattgc agcagccact ggctttgtca
      721 aaaaggacca gttgggcaag aatgaagaag gagccccaca ggaaggaatt ctggaagata
      781 tgcctgtgga tcctgacaat gaggcttatg aaatgccttc tgaggaaggg tatcaagact
      841 acgaacctga agcccatatg gctagcggcg gctctggtgg cggctctggt gtgagcaagg
      901 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      961 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1021 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1081 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1141 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1201 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1261 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1321 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1381 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1441 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1501 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1561 tgaccgccgc cgggatcact ctcggcatgg acgagctcta caagtgaaag cttcggaaga
     1621 agagccaggg cagcaagaat cctcagaagt actcggacaa ggagctgcag tacatcgacg
     1681 ccatcagcaa caagcagggc gagctggaga attacgtgtc cgacggctac aagaccgcac
     1741 tgacagagga gcgcaggcgc ttctgcttcc tggtggagaa gcagtgcgcc gtggccaaga
     1801 actccgcggc ctaccactcc aagggcaagg agctgctggc gcagaagctg ccgctgtggc
     1861 aacaggcctg tgccgacccc agcaagatct aagcggccgc ctcgagcacc accaccacca
     1921 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1981 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2041 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2101 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2161 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2221 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2281 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2341 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2401 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2461 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2521 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2581 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     2641 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2701 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2761 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2821 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2881 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2941 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     3001 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     3061 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     3121 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     3181 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     3241 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3301 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3361 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     3421 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3481 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3541 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3601 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3661 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3721 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3781 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3841 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3901 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3961 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4021 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4081 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4141 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4201 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4261 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4321 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4381 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     4441 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     4501 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4561 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4621 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4681 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4741 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4801 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4861 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4921 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4981 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5041 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5101 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5161 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5221 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     5281 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     5341 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     5401 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     5461 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5521 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5581 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5641 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5701 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5761 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5821 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5881 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5941 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     6001 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     6061 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     6121 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     6181 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     6241 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     6301 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     6361 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6421 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6481 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6541 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6601 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6661 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6721 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6781 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6841 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6901 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6961 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagagga
//
